The proposal aims to apply recombinant DNA technology to characterize the genetic and physiological basis of cyclophosphamide resistance in Lizio cells. Evidence is presented to indicate that the cyclophosphamide resistance is the consequence of an elevated aldehyde dehydrogenase activity. Using differential hybridization, we have cloned a candidate drug resistance gene. This gene has been used to clone cross-hybridizing chromosome fragments from a gene library prepared from the resistant cells. This gene will be sequenced in preparation for the production of an antibody against its gene product. The molecular basis for the differential expression in sensitive and resistant cells will also be determined. The effect of activated cyclophosphamide and related compounds on survival and DNA crosslinking in the recipient cells will be studied to determine that the general nature of the acquired resistance is related to an elevated aldehyde dehydrogenase activity. More detailed enzymological studies will seek to prove that the original resistance in L1210 cells and that acquired by gene transfer are identical phenomena.
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